一<em>线虫</em>研究Amylopathy模型系统
Summary
我们描述的方法来研究蠕虫amylopathies方面<em> C。线虫</em>。我们将展示如何构建表达Aβ的蠕虫<sub> 42</sub>在神经元和如何测试其功能行为分析。进一步的研究表明如何获取药理试验，可用于主培养的神经元。
Abstract
的Amylopathy是一个术语，描述了异常的β淀粉样蛋白（Aβ）的合成和积累组织中随着时间的推移。 Aβ是阿耳茨海默氏病（AD）的一个标志，并且被发现在路易体痴呆，包涵体肌炎和脑淀粉样血管病1-4。 Amylopathies随着时间的推移逐步发展。出于这个原因，简单的有机体寿命短，可能有助于阐明分子方面的这些条件。在这里，我们描述了实验协议利用线虫研究Aβ介导的神经退行性疾病。因此，我们构建转基因蠕虫通过注射DNA编码人类Aβ42到成年雌雄同体的合胞性腺。转化线是稳定的诱变诱发的整合。线虫是年龄同步收集和播种他们的鸡蛋。测试时机行为分析的功能神经元表达Aβ42（趋化实验S）。从胚胎的原代神经元培养物的使用行为数据的补充和测试抗凋亡的化合物的神经保护作用。
Introduction
β淀粉样蛋白（Aβ）是36-43个氨基酸的肽的顺序裂解后形成的淀粉样前体蛋白（APP），β和γ分泌1。的γ分泌的Aβ肽的C-末端进行处理，并负责可变长度5。 Aβ的最常见的形式是Aβ40和Aβ42，后者更常见的病理条件下，如AD 5相关联。 Aβ形式在高浓度的β-折叠，聚集形成淀粉样纤维6。存款的原纤维周围神经元的老年斑的主要成分。两个斑块和斑块扩散，非Aβ寡聚体，被认为构成了潜在的致病形式的Aβ。
实验室神经元amylopathies研究是复杂的事实，这些条件随时间的进展。因此，它是重要的吨至发展互补基因听话的动物模型小鼠寿命短。这些模型可以用来阐明具体方面amylopathies典型的细胞和分子，凭借其简单，有利于把握问题的实质。 线虫瀑布是这一类。它有一个很短的寿命，〜20天，除了基本的细胞过程，包括调节基因表达，蛋白质贩运，神经元连接，突触，细胞信号传导，和死亡的哺乳动物7类似。蠕虫的独特功能包括强大的遗传学和缺乏的血管系统，使独立研究神经元损伤血管损伤。另一方面，人脑的缺乏限制下使用线虫研究神经退行性疾病的许多方面。此外，病变的解剖分布的再现和识别不能进行这种微生物。其他限制ations包括两个基因表达谱的差异和复杂的行为和记忆功能的减值难以评估。在这里，我们描述的方法来生成C。线虫模型amylopathies。
Protocol
Representative Results
Discussion
在这里，我们描述相结合的办法，来研究细胞和分子方面，用C amylopathies 线虫 。这种方法的优点包括：1）成本低。 线虫保持在正常与细菌接种的陪替氏培养皿中，在室温下。 2）强大的遗传学。转基因动物在几个月内，可以得到各种各样的启动子序列可用来驱动在特定神经元的所需基因的表达。 3）操作简单，良好的特点，神经系统。C.线虫拥有一个非常简单的神经系?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Materials
| Name of Reagent | Company | Catalog Number | Comments |
| 1. NGM | |||
| Sodium Chloride | Sigma-Aldrich | S5886 | 3 g |
| Bacteriological agar | AMRESCO | J637 | 17 g |
| Bacto-peptone | AMRESCO | J636 | 2.5 g |
| Distilled Water | Bring to 975 ml | ||
| Sterilized by autoclaving, then add the following items and mix well | |||
| Magnesium sulfate | Sigma-Aldrich | M2643 | 1 ml of 1 M stock |
| Calcium Chloride | Sigma-Aldrich | C5670 | 1 ml of 1 M stock |
| Cholesterol | Sigma-Aldrich | C3045 | 1 ml of 5 mg/ml stock( in ethanol) |
| Potassium phosphate buffer | 25 ml of 1M stock | ||
| 2. Potassium phosphate buffer | |||
| Potassium phosphate monobasic | Sigma-Aldrich | P5655 | 108.3 g |
| Potassium phosphate dibasic | Sigma-Aldrich | P3786 | 35.6 g |
| Distilled Water | Bring to 1 L | ||
| Sterilized by autoclaving | |||
| 3. M9 buffer | |||
| Potassium phosphate monobasic | Sigma-Aldrich | P5655 | 3 g |
| Sodium phosphate dibasic | Sigma-Aldrich | S5136 | 6 g |
| Sodium Chloride | Sigma-Aldrich | S5886 | 5 g |
| Magnesium sulfate | Sigma-Aldrich | M2643 | 1 ml of 1 M stock |
| Distilled Water | Bring to 1 L | ||
| Sterilized by autoclaving | |||
| 4. Egg buffer (pH 7.3, 340 mOsm) | |||
| Sodium Chloride | Sigma-Aldrich | S5886 | 118 mM |
| Potassium Chloride | Sigma-Aldrich | P5405 | 48 mM |
| Calcium Chloride | Sigma-Aldrich | C5670 | 2 mM |
| Magnesium Chloride | Sigma-Aldrich | M4880 | 2 mM |
| HEPES | Fisher Scientific | BP310 | 25 mM |
| Distilled Water | Bring to 1 L | ||
| Sterilized by autoclaving | |||
| 5. CM-15 | |||
| L-15 culture medium | Gibco | 11415 | 450 ml |
| Fetal Bovine Serum | Gibco | 10437-028 | 50 ml |
| Penicillin | Gibco | 15140 | 50 units/ml |
| Streptomycin | Gibco | 15140 | 50 g/ml |
| Adjust to 340 mOsm with sucrose then sterile filter into autoclaved bottles and store at 4 °C | |||
| 6. Other Reagents | |||
| Halocarbon 700 oil | Halocarbon Products | 9002-83-9 | |
| 5 μm Acrodisc Syringe Filter | PALL Co. | 4199 | |
| Chitinase | Sigma-Aldrich | C6137-5UN | |
| Lectin (peanut) | Sigma-Aldrich | L0881-10MG | |
| Sodium hydroxide | Fisher Scientific | S320 | |
| Lysine | Sigma-Aldrich | L5501 | |
| Biotin | Sigma-Aldrich | B4639 | |
| Sodium hypochlorite solution | Sigma-Aldrich | 425044 | |
| Sodium azide | Sigma-Aldrich | 71289 | |
| Sucrose | Sigma-Aldrich | S0389 |
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